Eukaryotes have a highly conserved enzymatic system for the ligation of ubiquitin (Ub) to proteins, and often these proteins are then targeted for degradation by the proteasome. Substrates, include naturally short-lived regulatory factors and aberrant protein quality control (PQG) substrates. Many human disorders, including neurodegenerative diseases such as Alzheimer?s and Parkinson?s disease, diabetes, cystic fibrosis, and certain cancers, are associated with abnormalities in Lib-dependent proteolysis. The Ub-proteasome system presents promising drug targets for treating-these diseases. In this renewal, the PI proposes to extend studies On Ub-dependent proteolysis, focusing on endplasmic reticulum (ER)-associated degradation (ERAD) and basic features of membrane and nuclear protein ubiquitylation and degradation. The proposed research will focus on the yeast Saccharomyces cerevisiae because of its experimental advantages and the fact that the Ub system in general, and the ERAD machinery in particular, is highly conserved Recent work has Identified a yeast Ub-ligase (E3) complex embedded in the ER and nuclear envelope membranes that is capable of recognizing a wide array of regulatory and PQC substrates. This unusual complex includes a large integral membrane E3 called Doa10 and two Ub-conjugating enzymes (E2s), Ubc6 and Ubc7. Doa10 Is the prototype for a broadly conserved class of viral and eukaryotic Ub ligases. It was discovered from an analysis of a soluble nuclear substrate, the Mata2 transcription factor, but it also has membrane substrates. A second E3, Sxl5/Slx8, important for MATa2 degradation was also recently discovered. The overall goal of the proposal is to determine key mechanistic features of protein ubiquitylation by the ER-membrane E3 ligases DoalO and Hrd1. We also hope to advance our currently very poor understanding of how membrane extraction of ER membrane substrates occurs in conjunction with theseE3s. For the soluble substrate MAT ? 2, both its Doa10-dependent and Slx5/Slx8-dependent ubiquitylation will be explored. We expect to continue to gain important insights into fundamental aspects of Ub-proteasome system mechanism and function, including features unique to the PQC of membrane proteins at the ER.